Structure, function, regulation and expression of A -kinase anchor proteins

Abstract

Hormone-stimulated adenylate cyclase and ultimate downstream effectors can be separated by large distances in neurons and in highly polarized epithelial cells. Mechanisms by which diffuse cAMP signals are captured, amplified and focussed precisely on distal substrate-effector proteins are poorly understood. I have cloned a novel cDNA which encodes six distinct A Kinase Anchor Proteins (collectively named AKAP-KL) by alternative mRNA splicing and utilization of two translation initiation codons. AKAP-KL isoforms bind regulatory subunits (RII) of type II protein kinase A (PKA) and may be involved in transepithelial signal transduction. AKAP-KL isoforms are evident in lung, kidney and cerebellum, but are absent from many other tissues. Different isoforms predominate in different tissues. Mutagenesis revealed that several hydrophobic residues including Leu593Val594 and Ile598 and their precise positions within this RII binding site are crucial for the sequestration of RII. AKAP-KL proteins avidly ligate RII in vitro and in intact cells. The distribution of AKAP-KL protein is strikingly asymmetric (polarized) in situ. Anchor protein isoforms accumulate near the lumenal (apical) surface of the highly polarized epithelium of tubules in nephrons. AKAP-KL interacts with and modulates the structure of the actin cytoskeleton in transfected cells through a C-terminal coiled-coil domain. The properties of AKAP-KL suggest that this anchor protein may be involved in (a) establishing polarity in signaling systems and (b) physically and functionally integrating PKAII isoforms with downstream effectors in order to precisely target transepithelial signals carried by cAMP.;In neurons, class C L-type voltage-gated Ca2+ channel is a PKA effector protein. Phosphorylation by PKA increases the channel activity. A direct protein overlay binding assay performed with 32P-labeled RIIbeta revealed a prominent AKAP with an Mr of 280 kDa in immuno-isolated class C channel complexes. The protein was identified by immunoblotting as a previously-characterized AKAP, microtubule-associated protein 2B (MAP2B). In vitro GST-pull down assays show that MAP2B interacts with several cytoplasmic domains of the class C calcium channel.;Like AKAP-KL, AKAP75/79/150 expression is tissue-specific. AKAP75/79/150 is predominately expressed in brain according to Western and Northern blot results. Cloning and sequencing of genes and cDNAs encoding AKAP75/79/150 revealed that AKAP75/79/150 transcription unit has no introns in the coding region. However, three mRNA isoforms are generated by alternative splicing within the 5' end untranslated region of AKAP150 gene transcripts. The mouse AKAP150 gene has a TATA-less, GC-rich promoter with a single transcription initiation site for the three mRNA isoforms. A 5.6 kbp DNA fragment that precedes the initiator ATG has promoter activity in several cell lines. (Abstract shortened by UMI.).